Isolation and HPLC Quantitative Determination of 5α-Reductase Inhibitors from Tectona grandis L.f. Leaf Extract

Steroid 5α-reductase plays a crucial role in catalyzing the conversion of testosterone to dihydrotestosterone, which is involved in many androgen-dependent disorders. Leaf-hexane extract from Tectona grandis L.f. has shown promise as a 5α-reductase inhibitor. The objectives of this current study were to isolate and identify 5α-reductase inhibitors from T. grandis leaves and to use them as the bioactive markers for standardization of the extract. Three terpenoid compounds, (+)-eperua-8,13-dien-15-oic acid (1), (+)-eperua-7,13-dien-15-oic acid (2), and lupeol (3), were isolated and evaluated for 5α-reductase inhibitory activity. Compounds 1 and 2 exhibited potent 5α-reductase inhibitory activity, while 3 showed weak inhibitory activity. An HPLC method for the quantitative determination of the two potent inhibitors (1 and 2), applicable for quality control of T. grandis leaf extracts, was also developed. The ethanolic extract showed a significantly higher content of 1 and 2 than found in the hexane extract, suggesting that ethanol is a preferable extraction solvent. This study is the first reported isolation of 5α-reductase inhibitors (1 and 2) from T. grandis leaves. The extraction and quality control methods that are safe and useful for further development of T. grandis leaf extract as an active ingredient for hair loss treatment products are also reported.


Introduction
Testosterone is the major circulating androgen in many androgen-sensitive tissues that circulate androgen in the serum of men [1] and is ultimately synthesized by the Leydig cells of the testes under the control of the hypothalamus and anterior pituitary gland [2]. Testosterone can be metabolized in most tissues to dihydrotestosterone (DHT) by the enzyme steroid 5α-reductase where nicotinamide adenine dinucleotide phosphate (NADPH) is used as the cofactor [3]. The isoforms of human steroid 5α-reductase are distributed into three major isoforms (type 1-3) which are specifically located in human tissues [3,4]. The overproduction of DHT can cause several androgen-dependent disorders including androgenic alopecia (AGA), benign prostatic hyperplasia (BPH), prostate cancer, female hirsutism, and acne [5,6]. According to the literature, compounds 1 and 2 are naturally occurring and chemically prepared compounds. They were first isolated and identified as the chemical constituents in the MeOH extract of Sindora siamensis Miq. leaves [29]. Moreover, 1 has also been synthesized from sclareol by superacidic cyclization of alcohols [34][35][36] whereas 2 could be synthesized by MnO2 and AgNO3 oxidation of labda-7,13 E-diene-15-ol [37], while (−)−eperua-7,13-dien-15-oic acid, the enantiomer of 2, had been isolated from Hymenaea coubarril [31], Isodon scoparius [38], and Copaifera langsdorffii [39]. In addition, compound 3 was previously isolated from various plants such as T. grandis [33], Wrightia tinctoria R.Br. [40], Oxystelma esculentum (L. f.) Sm. [41], and Taraxacum officinale (L.) Weber ex F.H.Wigg. [42]. It should be noted that the present study described the isolation and identification of two eperuane-type diterpenes (1 and 2) as the chemical constituents in T. grandis for the first time.

Steroid 5-Reductase Inhibitory Activity
The steroid 5-reductase inhibitory activity of samples on the conversion of testosterone to DHT was determined using LNCaP cell as a source of enzyme. In the screening, the percentage of enzymatic inhibition of T. grandis leaf extracts and three isolated compounds were measured at the final concentration of 100 µ g/mL. All samples showed a steroid 5-reductase inhibitory effect greater than 80% inhibitory activity. Therefore, the concentrations that could inhibit 50% of enzymatic activity (IC50) of all samples were further determined. Two reference 5-reductase inhibitors, finasteride and curcumin, were determined using our assay system as described in Srivilai et al. (2017) [43]. Their IC50 values on 5-reductase are summarized in Table 1. Various classes of terpenoids in T. grandis leaves and bark have been previously reported, such as sesquiterpenes (eudesmane-and oppositane-types), diterpenes (phytaneand eperuane-types), and triterpenes (ursane-, oleanane-, and lupane-types) [20,33].

Steroid 5α-Reductase Inhibitory Activity
The steroid 5α-reductase inhibitory activity of samples on the conversion of testosterone to DHT was determined using LNCaP cell as a source of enzyme. In the screening, the percentage of enzymatic inhibition of T. grandis leaf extracts and three isolated compounds were measured at the final concentration of 100 µg/mL. All samples showed a steroid 5α-reductase inhibitory effect greater than 80% inhibitory activity. Therefore, the concentrations that could inhibit 50% of enzymatic activity (IC 50 ) of all samples were further determined. Two reference 5α-reductase inhibitors, finasteride and curcumin, were determined using our assay system as described in Srivilai et al. (2017) [43]. Their IC 50 values on 5α-reductase are summarized in Table 1. The results showed that inhibition of 5α-reductase by ethanolic extract (IC 50 = 23.91 ± 0.17 µg/mL) was more potent than hexane extract (IC 50 = 26.45 ± 0.69 µg/mL). Two isolated diterpenes (1 and 2) had the potent ability to inhibit 5α-reductase at IC 50 value of 1 of 14.19 ± 2.87 µM (or 4.31 ± 0.87 µg/mL) and, for 2, 14.65 ± 0.31 µM (or 4.45 ± 0.10 µg/mL). The 5α-reductase inhibitory activity of both 1 and 2 was significantly higher than a triterpene (3) but less than a standard 5α-reductase inhibitor, finasteride. Interestingly, there was no significant difference in the inhibitory activity between two potent compounds (1 and 2) and a positive control, curcumin. The limited number of compounds restricted the interpretation of structureactivity relationships. However, the presence of α,β unsaturated carboxylic acid in the side chain of 1 and 2 might be important for 5α-reductase inhibitory activity, while the less inhibitory activity of 3 is still largely unknown. Other factors might have been involved in the activity, which need to be investigated further. The importance of the carboxyl group for 5α-reductase inhibition has also been mentioned in the literature [44,45]. For example, ganoderic acid TR showed stronger inhibitory activity than 5α-lanosta-7,9(11),24triene-15α,26-dihydroxy-3-one. The only difference in these two compounds is the position of C-26 which is a carboxyl group for ganoderic acid TR, and the hydroxyl group for 5α-lanosta-7,9(11),24-triene-15α,26-dihydroxy-3-one. These results demonstrated that a carboxyl group of 17β-side chain of ganoderic acid TR is important to elicit the inhibitory activity. Meanwhile, the methyl ester of ganoderic acid TR showed much less inhibitory activity on 5α-reductase. Additionally, the presence of unsaturated at C-24 and C-25 of three most potent inhibitors (ganoderic acid TR, ganoderic acid DM, and 5α-lanosta-7,9(11),24triene-15α,26-dihydroxy-3-one) was imperative to their activity, while the fully saturated triterpenoids was less potent. The study of Srivilai et al. [46] also discussed the crucial role of α,β unsaturated ketone in sesquiterpenes for 5α-reductase inhibition.
For the pharmacological properties of these isolated compounds, 1 and 2 have been shown to possess histone deacetylase (HDAC) inhibitory activity [29], while 3 has shown anti-inflammatory activity [47], antimalarial activity [48], andapoptogenic activity [49,50], and to exhibit strong androgen receptor inhibitory activity [51]. Although the anti-androgenic effect via the androgen receptor inhibition by 3 had been reported, the 5α-reductase inhibitory activity of 1 and 2 had never been described before. The 5α-reductase activity of these compounds is reported here for the first time. For the further development of products containing T. grandis extract for medical or cosmetic applications, standardization and quality control are necessary to guarantee consistent levels of bioactive compounds in the extract. We then carried out a quantitative study of the two bioactive markers (1 and 2) in the extracts prepared from hexane, as the previous report suggested, and ethanol, which was more economic and environmentally friendly.

Quantitative HPLC Analysis of 5α-Reductase Inhibitors from T. grandis Leaf Extract
The HPLC method for the quantitative determination of the two active compounds 1 and 2 was developed and validated according to ICH guidelines. The wavelength for quantitative determination at 220 nm was chosen to obtain the baseline separation of 1 and 2 when used as markers.
An isocratic elution of acetonitrile-formic acid in purified water as the mobile phase was conducted to successfully separate compounds 1 (t R 14.52 min) and 2 (t R 13.15 min) in the hexane and ethanolic extracts of T. grandis leaf within 15 min ( Figure 2). The results of the method validation parameters for the determination of 1 and 2 are summarized in Table 2.
Molecules 2022, 27, 2893 5 of 12 quantitative determination at 220 nm was chosen to obtain the baseline separatio and 2 when used as markers.
An isocratic elution of acetonitrile-formic acid in purified water as the mobile was conducted to successfully separate compounds 1 (tR 14.52 min) and 2 (tR 13.15 m the hexane and ethanolic extracts of T. grandis leaf within 15 min ( Figure 2). The res the method validation parameters for the determination of 1 and 2 are summari Table 2.  0.09 µ g/mL 0.06 µ g/mL Limits of quantification (LOQ) 0.30 µ g/mL 0.20 µ g/mL As a result, the plot of peak area versus the concentrations (1.56-200 µ g/mL) o 2 provided good linearity for this method, with r 2 of 0.9997 for 1 and 0.9995 for 2. Th of 1 was 0.09 µ g/mL and 2 was 0.06 µ g/mL, while the LOQ of 1 was 0.30 µ g/mL and 0.20 µ g/mL, indicating a high sensitivity of the method.
The analytical method developed for the quantification of 1 and 2 had good acc with the overall recovery in the range of 92.78-100.6%. The RSD values were less th for the intra-day and inter-day which demonstrated the high precision of the metho ble 3). These results showed that the developed quantitative method was sensitive rate, and precise to determine two active constituents in the T. grandis leaf-extracts taneously.  As a result, the plot of peak area versus the concentrations (1.56-200 µg/mL) of 1 and 2 provided good linearity for this method, with r 2 of 0.9997 for 1 and 0.9995 for 2. The LOD of 1 was 0.09 µg/mL and 2 was 0.06 µg/mL, while the LOQ of 1 was 0.30 µg/mL and 2 was 0.20 µg/mL, indicating a high sensitivity of the method.
The analytical method developed for the quantification of 1 and 2 had good accuracy, with the overall recovery in the range of 92.78-100.6%. The RSD values were less than 3% for the intra-day and inter-day which demonstrated the high precision of the method (Table 3). These results showed that the developed quantitative method was sensitive, accurate, and precise to determine two active constituents in the T. grandis leaf-extracts simultaneously. Table 3. Accuracy (% recovery) and intra-and inter-day precisions of 1 and 2 by the proposed HPLC method.

RSD (%)
Intra-Day a Inter-Day b The contents of the two 5α-reductase inhibitors, 1 and 2, in hexane and ethanolic extracts of T. grandis leaf, were investigated using our validated HPLC method, previously discussed (see Tables 2 and 3 for details). The peak identification of these components was characterized by comparison with the retention time of the reference compounds (Figure 2c). The results revealed that the ethanolic extract exhibited significantly higher contents of 1 (6.18 ± 0.12 % (w/w) and 2 (3.83 ± 0.04 % (w/w), while the hexane extract contained 5.60 ± 0.05 % (w/w) of 1 and 3.23 ± 0.03 % (w/w) of 2 (Table 4). This is in agreement with the fact that higher 5α-reductase inhibitory activity was found in the ethanolic extract. In  Moreover, ethanol is safe and has wide acceptability as an extraction solvent and ingredient in food, drugs, and cosmetics [52][53][54]. Therefore, 95% ethanol is recommended as a solvent for extracting T. grandis leaves with the further application as an ingredient in the drugs and cosmeceutical products for hair-loss treatment.

General Experimental Procedures
Thin layer chromatography (TLC) analysis was performed on TLC silica gel 60 F254 aluminum sheet 20 × 20 cm (Merck, Darmstadt, Germany). A silica gel column (0.040-0.063 mm granule size), a sephadex LH-20 column (particle size dry 18-111 µm), and a C18 column (40-63 particle size) were used for chromatographic isolation of the extract constituents. Fouriertransform infrared (FT-IR) spectra were recorded with attenuated total reflectance (ATR) mode on a PerkinElmer spectrum GX (Perkin Elmer, Waltham, MA, USA). Optical rotations were measured using a POLAX-2L polarimeter (Atago, Japan). An Agilent 1260 infinity high performance liquid chromatography instrument via an electrospray ionization (ESI) interface to a 6540 ultrahigh definition accurate mass Q-TOF (Agilent Technologies, Palo Alto, CA, USA) was conducted. Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker AV400 (Bruker, Billerica, MA, USA) spectrometer at 400 MHz for proton and 100 MHz for carbon. The absorbance was measured using hybrid Multi-Mode Detection Synergy H1 (Model H1MF) (Bio-TeK Instruments, Winooski, VT, USA). The high performance liquid chromatography (HPLC) was performed using Agilent Technology (model 1260 infinity with fraction collector, Santa Clara, CA, USA).

Plant Material
Fresh mature leaves of T. grandis were collected from Banna district, Nakhon Nayok Province, Thailand in September 2019. The plant material was identified by Assist. Prof. Dr. Pranee Nangngam, Faculty of Science, Naresuan University. The voucher specimen (collection no. 05721) was deposited at the Department of Biology, Faculty of Science, Naresuan University, Phitsanulok, Thailand.

Extraction and Isolation
The fresh mature leaves of T. grandis (TG) were chopped into small pieces and dried at 55 • C. The dried material was ground into a fine powder and passed through a 60-mesh sieve. The T. grandis leaf powder was extracted individually using two organic solvents; hexane and 95% ethanol. For the preparation of crude hexane extract, the T. grandis leaf powder (1.5 kg) was macerated with hexane (6.0 L) three times (at least five days each time) at room temperature with occasional shaking, and the solvent was removed under reduced pressure to produce a dark green viscous crude hexane extract (127 g, 8.47% yield). For the preparation of crude ethanolic extract, a 292 g sample of the leaf powder was macerated with 95% ethanol (1.17 L) and, following the same procedure previously described, a dark green viscous crude ethanolic extract (32.90 g, 11.27% yield) was produced. Both of the resultant crude extracts were stored at −20 • C until used.
(+)-Eperua-8,13-dien-15-oic acid (1) by computer matching its recorded mass spectrum with the Wiley7n standard library and with data found in the literature.

Enzyme Preparation
5α-reductase was prepared as a crude enzyme from the androgen-dependent LNCaP cells (ATCC ® CRL-1740 TM ) using the procedure as described by Fachrunniza et al. [28], which was modified from Srivilai et al. [55]. Briefly, the LNCaP cells were cultured in a 175 cm 2 culture flask at 37 • C under a 5% CO 2 humidified atmosphere and cultured until reaching approximately 80% confluency. The medium was removed and the cells were rinsed with tris-HCl buffer solution, pH 7.4 (containing 10 mM Tris-HCl buffer; 50 mM KCl; 1 mM EDTA; 0.5 mM phenylmethanesulfonyl fluoride). The cells were then scraped off and centrifuged at 1900× g for 10 min. The cell pellets were collected and re-suspended in tris-HCl buffer solution pH 7.4 to a concentration of ≥9 × 10 7 cells/mL and the resultant cell pellets were kept in an ice bath and homogenized using a sonication probe. The total protein content in the homogenized crude enzyme was not less than 75 µg protein equivalent in this 5α-reductase inhibitory assay, which was measured using the Pierce bicinchoninic acid (BCA) protein assay (Pierce, Rockford, IL, USA).

Enzymatic 5α-Reductase Inhibition Assay
The in vitro inhibitory activity of the samples against the conversion of testosterone to DHT by 5α-reductase was carried out according to the method described by Srivilai et al. [55]. The DHT formation after the enzymatic reaction was determined using liquid chromatography mass spectrometry (LC-MS) to measure 5α-reductase activity. Curcumin and finasteride, which have been reported as 5α-reductase inhibitors [43,56], were used as the positive controls. In the assay, the reaction was performed in U-shaped 96-deepwell plates covered by well-cap mats to create a solution that contained 10 µL of the tested sample dissolved in dimethyl sulfoxide (DMSO), 20 µL of testosterone (34.7 µM in propylene glycol and water), and 50 µL of β-nicotinamide adenine dinucleotide phosphate (NADPH, 1 mM in tris-HCl buffer pH 7.4). The enzymatic reaction was started by adding 80 µL of homogenized crude enzyme (equivalent to 75 µg protein), and the final volume was adjusted to 200 µL by adding 40 µL of tris-HCl buffer pH 7.4. The reaction was maintained in a water bath with a shaker at 37 • C for 60 min and then the reaction was stopped by adding 300 µL of hydroxylamine hydrochloride (HM) (10 mg/mL in 80% v/v ethanol). The solution was then incubated for another 60 min at 60 • C to completely derivatize all the DHT that was produced. After incubation, the solution was centrifuged at 1700× g for 10 min and the supernatant was collected for quantitated DHT production by LC-MS. Two control groups, C 0 and C 60 , were prepared with all the solutions, including 10 µL of DMSO, but no test sample. Control group C 0 was stopped (by adding HM) before the enzymatic incubation at 0 min, while the control group C 60 continued with the enzymatic reaction and was stopped after 60 min of incubation. The DHT production was determined as the diluting solvent to achieve the desired concentration. All standard solutions were filtered through a 0.45 µm nylon membrane before being injected into the HPLC system.

Chromatographic Conditions
The HPLC apparatus was an Agilent 1260 infinity equipped with a G1315D HPLC diode array detector (Agilent Technologies, Santa Clara, CA, USA). The chromatographic separation was performed on a reversed-phase Phenomenex Luna C18(2) column (150 mm × 4.6 mm, 5 µm particle size). A mixture solution of acetonitrile and 0.1% (v/v) formic acid in purified water (85:15 v/v) was used as the mobile phase. The isocratic elution system was programmed with a 0.8 mL/min flow rate at room temperature and the UV chromatogram was recorded at 220 nm. The injection volume was 20 µL.

Validation of HPLC Method
According to the International Council for Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) guidelines [57], the HPLC method that we developed was validated, including the linearity, limit of detection (LOD), the limit of quantification (LOQ), accuracy, and intra-day and inter-day precision. The linearity range of 1 and 2 were determined on eight concentration levels (1.56-200 µg/mL) with triplicate experiments. A calibration curve was created by plotting the mean of the peak areas versus their concentrations and expressed by calculating the slope, y-intercept, and the squared regression coefficient (r 2 ). LOD and LOQ under the present chromatographic conditions were calculated using the spiked sample blank method applying the lowest known concentration of standard solutions, calculated in 10 replicates. The LOD and LOQ were determined by calculating the standard deviation of the response (which are usually represented as the concentration of analytes in the sample) based on the signal to noise ratio (S/N) equal 3 for LOD and 10 for LOQ. The accuracy of the method was determined using the spiked sample method. Three different concentration levels of 1 and 2 mixtures (15, 75, and 135 µg/mL) were added to the crude ethanolic extract solution covering the specified range in 25, 50, and 75% of their calibration curve. These experiments were completed in triplicate. The accuracy is expressed as a percentage of recovery, which was calculated from 100 × [(C spiked − C non-spiked )/C standard ], where C is concentration in µg/mL unit.
The precision of the method was verified by repeatability (intra-day precision) and intermediate precision (inter-day precision) studies. These studies were performed by analyzing three concentration levels (20,75, and 150 µg/mL). Intra-day precision was determined by three replicated analyses of each concentration within 1 day (n = 3). Interday precision was determined in triplicate for consecutive three days. Precision was expressed as a percentage of relative standard deviation (%RSD) calculated from the (standard deviation/mean × 100). For quantification of the two markers, 1 and 2 in T. grandis extracts were calculated from the corresponding calibration curve.

Statistical Analysis
Data were expressed as the means ± standard deviation (SD) of at least triplicate experiments. Statistical comparisons were analyzed using one-way analysis of variance (ANOVA), followed by Duncan's test. p < 0.05 was considered statistically significant.

Conclusions
This study has shown, for the first time, the isolation and identification of 5α-reductase inhibitors; two eperuane-type diterpenes (1 and 2) and a lupane-type triterpene (3) from the leaf extract of T. grandis. The HPLC analysis of the two potent 5α-reductase inhibitors, 1 and 2, were developed, validated, and successfully applied for these compounds in T. grandis leaf extracts. The extraction of T. grandis leaves with 95% ethanol resulted in a higher volume of extract yielded which contained higher concentrations of 1 and 2, as well as exhibiting greater 5α-reductase inhibitory activity, than the hexane extract. Our discovery suggests that 1 and 2 can be bioactive markers for the further development of T. grandis leaf extract as an ingredient in the cosmeceutical products for hair loss treatment.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.